This project is intended to provide information pertaining to the control of enzymatic activity in urocanase and threonine dehydratase, two key enzymes concerned with amino acid catabolism in all organisms. Urocanase from Pseudomonas putida has been found to contain a new prosthetic group, alpha-ketobutyrate, whose function is to facilitate the addition of water in urocanate to produce imidazolone propionate. Of principal interest is the origin of this carbonyl coenzyme on the enzyme protein. Threonine is an immediate precursor of the alpha- ketobutyrate moiety, and mutants lacking the enzyme threonine dehydratase also lack the ability to form active urocanase. In this project, the possible existence of a proenzyme form of urocanase, lacking alpha-ketobutyrate, will be explored in the mutants lacking threonine dehydratase. Alternatively, should such a proenzyme form not be found, the possibility will be considered that alpha-ketobutyrate is formed from peptide-bound threonine by threonine dehydratase at a stage of urocanase biosynthesis prior to release from ribosomes. Another aspect of this work will be concerned with the elucidation of the nature of the coenzyme of urocanase from guinea pig liver, and its possible biosynthesis. Threonine dehydratases from Clostridium tetanomorphum and P. putida are being investigated from several points of view. Of primary concern in the former is the relationship between quaternary structure and control of the enzymatic activity by 5'-ADP. A combination of ultracentrifugal studies, fluorescence analysis, ligand binding measurements and covalent modification studies will be employed to answer the question of whether oligomerization is obligatorily linked to the activation of the enzyme by ADP. The threonine dehydratase of P. putida has been implicated in the biosynthesis of urocanase. However, it also has an unusual coenzyme replacing the normal pyridoxal phosphate. The nature of this coenzyme will be investigated and studies initiated on its biosynthesis.